Anti actin a4700

Whole-cell extracts were prepared by the direct addition of 1 × Laemmli sample buffer (62.5 mM Tris [pH 6.8], 2% SDS, 10% glycerol, 0.02% bromophenol blue, 5% 2-mercaptoethanol) to the cells in the culture plate. Cells were scraped with a cell scraper (TPP, Trasadingen, Switzerland), and lysates were collected then incubated at 95 °C for 5 min. Cell extracts were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using 12% polyacrylamide gels. Proteins were transferred onto a nitrocellulose membrane (Whatman, Kent, UK) by electroblotting at 150 mA for 90 min on ice in a transfer buffer comprising 25 mM Tris, 192 mM glycine, and 20% isopropanol. Ponceau staining was used to assess the efficacy of the transfer and to facilitate cutting the membrane when probing the same membrane with multiple antibodies. Primary antibodies (anti-TOP1 ab109374 Abcam; anti-MITF HPA003259 Cambridge Bioscience Ltd; anti-vinculin V9131 Sigma; anti-actin A4700 Sigma) were incubated with membranes overnight at 4 °C with agitation. Protein bands were detected by an enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). The bands’ intensity was semi-quantified using the software ImageJ.